The present invention relates to nucleic acid sequences from Xenorhabdus nematophila. The invention also relates to methods of using the disclosed nucleic acid molecules to encode proteins and fragments of proteins and to develop antibodies, for example, for nucleic acid sequence identification and analysis, preparation of constructs, transformation of cells such as bacterial cells and plant cells with the nucleotide compositions disclosed herein to produce Xenorhabdus proteins or fragments thereof, in particular novel insect inhibitory, bactericidal, fungicidal and nematocidal proteins.
Xenorhabdus is a Gram-negative bacterium, a member of the family of Enterobacteriaceae, and symbiotically associated with nematodes of the genus Steinernema. The nematode-bacterial complex can be characterized as an obligate and lethal parasitic relationship, specializing in parasitizing and proliferating in soil insect larvae. Infective, non-feeding stages of these nematodes live in soil and carry in their gut the nematode-genus-specific symbiotic bacteria. It is believed that the nematodes actively search for the appropriate insect host, invade the insect larvae through natural openings or lesions in the cuticle and, once inside the hemolymph, release their symbiotic bacteria. The nematode-bacterial complex secretes a variety of highly efficient extracellular metabolites and proteins exhibiting insecticidal, bactericidal, fungicidal and nematocidal properties to secure the larval mass as a source of nutrition. An array of extracellular enzymes such as lipases, phospholipases, proteases, nucleases as well as several broad spectrum antibiotics, and antifungal and nematocidal compositions are also secreted [Boemare & Akhurst, J. Gen. Microbiol. 134: 751-761, 1988; Li et al., Can. J. Microbiol. 43(8):770-773, 1997; McInerney et al., J. Nat. Prod. 54(3):774-84, 1991; McInerney et al., J. Nat. Prod. 54(3):785-95, 1991; Sundar and Chang, J. Gen. Microbiol. 139 (Pt 12):3139-48, 1993]. It has been discovered that some compounds secreted by Xenorhabdus exhibit anti-neoplastic (U.S. Pat. No. 5,827,872), acaricidal, anti-inflammatory and anti-ulcerogenic properties (U.S. Pat. No. 4,837,222). U.S. Pat. No. 6,048,838 describes insect inhibitory proteins that exhibit a molecular weight of greater than 100 kDa produced by Xenorhabdus sp., which are active against a variety of insect species including the orders, Lepidoptera, Coleoptera, Diptera, and Acarina, when provided in the insect diet.
Xenorhabdus strains have been shown to produce an array of extracellular proteins and small molecules or secondary metabolites having specialized functions [Li et al., Can. J. Microbiol. 43(8):770-773, 1997; McInerney et al., J. Nat. Prod. 54(3):774-84, 1991; U.S. Pat. No. 6,048,838]. More commercially interesting are proteins and small molecules having antibiotic properties or proteins that exhibit insect inhibitory activity. A small number of insect inhibitory proteins have previously been identified from these bacteria, symbionts of insect-parasitic nematodes (Morgan et al., Appl. Environ. Microbiol., 67(5):2062-2069, 2001; U.S. Pat. No. 6,048,838). Such proteins and compositions are used as biologically safe and effective pest control agents. Unlike chemical pesticide compositions, these proteins have no effect upon the environment in general, can be targeted to direct their effect primarily upon target insect species, and have no effect on non-target species. Therefore, a different resistance management strategy that takes advantage of insect inhibitory proteins derived from distinct microbial sources other than B. thuringiensis would be desirable. Insect inhibitory proteins isolated from Xenorhabdus bacteria seem to have all the prerequisites for the delivery of novel genes for transgenic expression of insect pest inhibiting proteins to provide pest resistance to plants, either alone or in combination with Bacillus thuringiensis insecticidal proteins.
Therefore, there is a great deal of interest in identifying the genes that encode new insect inhibiting proteins, and proteins involved in the biosynthetic pathways of novel antibiotics produced by Xenorhabdus bacteria, as well as other useful proteins. Isolation and sequencing of the entire genome of Xenorhabdus would facilitate such an endeavor, because it would allow dissection and analysis of the genome into discrete genes encoding proteins having beneficial properties as described herein.